NUT Midline Carcinomas in the Thymic Region Yesim Go¨Kmen-Polar1, Oscar D Cano1, Kenneth a Kesler2, Patrick J Loehrer3 and Sunil Badve1,3

NUT midline carcinomas in the thymic region Yesim Go¨kmen-Polar1, Oscar D Cano1, Kenneth A Kesler2, Patrick J Loehrer3 and Sunil Badve1,3

1Departments of Pathology and Laboratory Medicine, Indiana University School of Medicine and Indiana University Simon Cancer Center, Indianapolis, IN, USA; 2Department of Surgery, Indiana University School of Medicine and Indiana University Simon Cancer Center, Indianapolis, IN, USA and 3Department of Internal Medicine, Indiana University School of Medicine and Indiana University Simon Cancer Center, Indianapolis, IN, USA

NUT midline carcinomas (NMCs) are rare tumors described predominantly in the pediatric age group. We recently reported two cases of these tumors occurring in the thymic region. In order to establish the true incidence of these tumors, we examined a large series of thymic carcinomas for morphological features of NUT tumor and further assessed the expression of NUTM1 (also known as NUT) protein by immunohistochemistry. The histological review of slides from 110 cases of thymic carcinoma was undertaken to identify carcinomas with mixed undifferentiated and squamous features that are typically associated with NUT carcinomas. The presenting symptoms, morphological spectrum of tumors and outcome data of patients with these histologies are presented. Immunohistochemistry for NUTM1 was performed on 35 cases of thymic carcinoma with available blocks (3 with these histological features and 32 without these features) to exclude the possibility of midline carcinoma. Tumors from 10 patients had features of mixed small cell undifferentiated squamous cell carcinoma (M:F, 1.5:1; age range, 22–79). These patients predominantly presented with advanced disease and had respiratory-related symptoms or chest pain; four had paraneoplastic syndromes. The squamous component in all cases was well differentiated with little or no atypia. The undifferentiated component varied in cell size and lacked characteristic features of small cell carcinoma. All but one patients developed metastases or died within 3 years of diagnosis. NUTM1 expression was seen in two of three tumors with these histological features and in none of the 32 cases without. Mixed small cell undifferentiated carcinomas share histological and immunohistochemical similarity with NMCs and have aggressive clinical course. These tumors are not uncommon and should be considered in the differential diagnosis of carcinomas in the thymic region as novel therapies might be available. Modern Pathology (2014) 27, 1649–1656; doi:10.1038/modpathol.2014.63; published online 23 May 2014

Keywords: mixed small cell carcinoma; NMC; NUT midline lethal carcinoma; thymus neoplasms; undifferentiated carcinoma

Thymic carcinoma is a rare malignant epithelial subtypes that can be indistinguishable from other tumor derived from the thymic gland in the anterior poorly differentiated carcinomas such as nuclear mediastinum. The disease is characterized by protein in testis (NUT) midline carcinomas cytological atypia and by invasiveness presenting (NMCs).8 NMCs, also called carcinoma with high risk of relapse and death.1–3 The current World t(15;19) translocation, are characterized by chromo- Health organization (WHO) schema recog- somal rearrangement of the NUTM1 gene located on nizes at least 11 histological subtypes. Combined chromosome 15 (commonly called NUT).9 NMCs are mixed tumors, like neuroendocrine carcinoma– histologically composed of an admixture of small thymic carcinoma, have also been reported.4–7 cell and undifferentiated carcinoma-type tumor Undifferentiated thymic carcinoma is one of the cells. The pattern is not dissimilar to that described for a rare variant of thymic carcinoma under the rubric of ‘mixed small cell undifferentiated 6 Correspondence: Dr S Badve, MD, FRCPath, Department of squamous cell carcinoma’ by Snover et al. They Pathology and Laboratory Medicine, Indiana University School described three cases of primary thymic carcinoma, of Medicine and Indiana University Simon Cancer Center, 350 wherein the tumor showed a mixture of well- West 11th street, IUHPL 4050, Indianapolis, IN 46202, USA. E-mail: [email protected] differentiated squamous carcinoma with a small Received 31 December 2013; revised 27 February 2014; accepted 6 cell undifferentiated component. Macroscopically, March 2014; published online 23 May 2014 the tumors were associated with gross evidence of www.modernpathology.org NUT midline carcinomas 1650 YGo¨kmen-Polar et al

invasion and necrosis. Microscopically, the squa- istry for NUTM1 protein (Cell Signaling, Danvers, mous cell component was described as discrete foci MA) in the three cases with available blocks. of well-differentiated squamous cell carcinoma with All cases included in this report were selected focal keratinization and occasional mitotic figures, based on the basis of hematoxylin and eosin-stained embedded in a sheet of an undifferentiated compo- morphology. The selection criterion was the nent. This undifferentiated component was similar to presence of two distinct components, namely an classical small cell tumor of lung and showed nests undifferentiated cell component and a moderate to of small cells delineated by delicate fibrous septae, well-differentiated squamous cell component, with- scant cytoplasm, finely granular nuclear chromatin, in the same tumor. The undifferentiated component high mitotic count, and extensive areas of necrosis. In was further classified as small or intermediate one of these three cases, undifferentiated compo- or large based on the criteria used in lung tumors. nent was described as showing areas where tumor Each case was analyzed for predominant architec- cells were larger, with vesicular nuclei and promi- tural pattern and cell type of the undifferentiated nent eosinophilic nucleoli. Delimitation of squa- component. The tumors were further classified as mous undifferentiated component was described as having organoid growth pattern defined as nested or abrupt with almost no transition between. Clini- trabecular growth pattern with perilobular palisad- cally, all three tumors showed an aggressive beha- ing, and associated with relatively scant stroma; or vior, unresponsiveness to treatment and associated desmoplastic growth pattern consisting of nest and with fatal outcome in 1–5 years. cords with broad zone of hyaline fibrous stroma, More recently, Mukai et al10 published a case with or without lymphoplasmacytic infiltrate. The of invasive thymic carcinoma with a mixed small percentage of squamous cell component was eval- cell and squamous cell component. In contrast uated as ‘low’ if presented in less than 50% of tumor to cases reported by Snover et al,6 the squamous and ‘high’ if presented in more than 50% of tumor. cell component in this case was described as being Mitotic figures were separately counted in both the ‘undifferentiated’ on histological and immuno- components and mitotic activity was classified as histochemical examinations. In spite of aggressive high (Z5 mitotic figures per 10 high power field treatment, including chemotherapy, radiotherapy and (hpf)), moderate (41/10 hpf but less than 5), or low radical surgery, the patient died 3 years after (r1/10 hpf). Intratumoral lymphocytic infiltrate diagnosis. and percentage of necrosis were also recorded. The goal of the current study was to identify the The demographic and clinical data collected incidence of mixed small cell undifferentiated included age, sex, race, history of smoking, date of squamous carcinoma of the thymus and examine initial diagnosis, presenting feature, stage of diag- its relationship with NMCs. Our review identified nosis, associated paraneoplastic/autoimmune syn- 10 cases of thymic carcinoma that exhibited this dromes, and surgical findings. Details about histology. Herein, we describe the presenting symp- systemic chemotherapy and radiation therapy were toms, morphological spectrum and outcomes data recorded. Follow-up information with respect to of these patients. NUTM1 expression in the tumors development of metastases, site of metastases, and was confirmed in two of the three cases on which duration of follow-up was extracted from patient blocks were available. charts. Survival information was obtained from charts, tumor registry, and by search of the death registry by social security number and date of birth. Materials and methods Immunohistochemical analysis for NUTM1 protein was performed on 35 cases where blocks or unstained Following the approval of the Institutional Review slides were available (3 cases of mixed histology and Board, we retrospectively reviewed the database at 32 cases without histological features as control). Indiana University. All the cases were diagnosed as Staining was performed using the C52 antibody (Cell malignant thymic neoplasms based on their clinical Signaling) and methods described by French et al.11 history, presenting symptoms and findings of The NUTM1 (C52B1) rabbit mAb detects endogenous imaging and histological studies. All patients levels of total NUT protein corresponding to the were referred to Indiana University Simon Cancer NUTM1 gene (NUT midline carcinoma, family Center for management of their primary or meta- member 1). The antibody also detects endogenous static thymic carcinomas. All available slides were levels of the BRD4-NUT fusion protein present in reviewed. Unstained slides or blocks were available NUT midline carcinoma, family member 1. for only three cases (in spite of contacting the Briefly, 5-mm sections, following deparaffinization submitting institutions); this prevented systematic and rehydration, were subjected to antigen retrieval study of the immunohistochemical findings. Immu- in Dako pH 9.0 solution (DakoUSA, Carpinteria, CA) nohistochemistry data, gross description and initial using Decloaking Chamber (BioCare Medical, diagnosis, where available, were collected from Walnut Creek, CA). After blocking and quenching archival pathology reports from original institution. endogenous peroxidase activity, sections were incu- The possibility of NUT-associated midline carcino- bated with primary rabbit monoclonal anti-NUT anti- ma was assessed by performing immunohistochem- body, and, after washes, incubated with horseradish

Modern Pathology (2014) 27, 1649–1656 NUT midline carcinomas YGo¨kmen-Polar et al 1651 peroxidase-conjugated secondary antibodies (Envision detection kit, DakoUSA). Staining was developed through incubation with diamino- benzidine, and sections were counterstained with hematoxylin. (months) Outcome Results Follow-up Clinical Findings The review identified 10 patients with mixed small Response cell undifferentiated squamous cell carcinoma; their to therapy clinical characteristics are summarized in Table 1. RT N/A 3 Dead RT PD 16 Dead RT PR 6 Dead RT PD 31 Dead non-small carcinoma with neuroendocrine þ þ þ Six of these patients were male (and four female) þ ge cell undifferentiated type with squamous CT CT CT CT PR 7 Dead CT PD 8 Dead with an average age at diagnosis of 50.9 years (range, CT þ þ þ þ þ 22–79). Two patients were Caucasians, two African þ Americans, one Asian, and one Middle Eastern; race information was not available in four patients. One of the patients had a prior history of prostate cancer (Case 9) and another had received radiation as a PDTC S Initial diagnosis Therapy PDTC N/A N/A 2 Dead PDTC S child for enlarged adenoids (Case 3). Five patients had significant history of smoking. Presenting symptoms were recorded for eight patients; four patients presented only with chest symptoms such as shortness of breath, cough and/or chest pain and soft tissue and liver whereas four presented only with systemic symp- lymph node toms such as weight loss, fatigue, and lymphadeno- pathy. Two patients presented with both chest and systemic symptoms. Four patients had symptoms Stage at diagnosis Metastasis IVb Bone and CNS U-SCC S IVa Pleura PDTC S related to autoimmune disease at presentation; one N/A N/A NSC-NE S each with Guillain–Barre´ syndrome, collagen vas- cular disease (Sjo¨gren syndrome and dermatomyositis), carcinoid-like syndrome, fibromyalgia and pancytopenia. Signs and symptoms suggestive syndrome ´ of possible paraneoplastic syndromes documented in the follow-up for four patients included hyponatremia, flushing, pancytopenia, clubbing, skin gren syndrome and rash and fibromyalgia. ¨ Fibromyalgia and pancytopenia UnknownGuillain–Barre IVb N/A WDTC CT PD N/A N/A dermatomyositis Rash and facial flushing IVb Lymph node TC CT PD 28 Dead

Stage and Metastases

In all cases, the tumors were diagnosed as thymic þ carcinoma. Tumor stage at diagnosis had been recorded for only eight patients. Five were tumor stage IVb, two were stage IVa, and one was stage IIb. spasms, and bronchitis and fatigue Surgical excision was performed in six cases. lymph node Extensive disease, in the form of diffuse pleural dissemination (Case 7) or presence of metastases at diagnosis (five cases), was documented in six cases. Metastatic involvement of two or more sites, Smoking history Presenting symptoms Paraneoplastic features including lymph nodes (n ¼ 4), brain (n ¼ 2), bones (n ¼ 2), liver (n ¼ 1) and abdominal wall (n ¼ 1), was noted in these cases. One patient developed soft tissue metastases 14 months after the initial diagnosis (Case 10).

Therapy Information The clinical data of the 10 patients with thymic mixed small cell undifferentiated squamous cell carcinoma Therapy information was documented in eight cases. Table 1 Case no. Age Sex Race 10 54 F As N/A Cough Unknown IVa Lymph node Abbreviations: AA, African American; C,feature; Caucasian; CNS, PD, central progressive nervous system; disease; CT, chemotherapy; PDTC, ME, poorly Middle Eastern; differentiated N/A, squamous not available; carcinoma; NSC-NE, PR, partial response; RT, radiotherapy; S, surgery; U-SCC, lar differentiation; WDTC, well-differentiated thymic carcinoma. 9 55 M ME Yes Weight loss Unknown N/A N/A PDTC S 8 37 F C Yes Back pain, cough, 4 39 M N/A N/A N/A Unknown IVb Lymph node 5 62 M N/A Yes Chest discomfort 1 79 M C N/A Left supraclavicular 67 41 61 M F N/A AA N/A Yes N/A Chest pain Hyponatremia, Unknown IIb N/A WDTC N/A N/A 3 N/A 2 22 F AA N/A Dyspnea, chest pain Unknown IVb Brain, bone, Two tumors were unresectable at the time of 3 59 M N/A Yes Cough Sjo

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diagnosis and these patients (Cases 1 and 5) received relatively abrupt with the two components being chemotherapy only. Six patients underwent surgery separated by one to three cells of intermediate followed by chemotherapy alone (Cases 3 and 7) morphology (Figure 1). The pathological findings, or by chemotherapy and radiotherapy (Cases 2, 8, 9, including the number of hematoxylin and eosin- and 10). Five patients had limited or no response to stained slides available for each reviewed case therapy and their condition continued progressing. (range, 1–14), are summarized in Table 2. Intratu- Two patients (Cases 7 and 8) had a partial response moral necrosis was observed in four cases, although (450% regression) to therapy. For the remaining in additional two cases, areas of necrosis were three patients (Cases 4, 6, and 9), neither the type of described on gross examination. Nine cases showed therapy nor their response was available. desmoplastic growth pattern whereas one case showed organoid growth pattern. Focal scant to moderate intratumoral lymphocytic infiltrate was Survival Information present in five cases. The undifferentiated component consisted of sheets In spite of surgical, chemotherapy and radiotherapy of cells arranged in syncytial growth pattern with treatment, the course of the disease was aggressive occasional areas exhibiting a discohesive architecture. with fatal outcome for eight patients ranging from 2 In four cases, a mixture of classic small and larger to 31 months following initial diagnosis (median, 7 intermediate cells was noted. In most cases, small months). For the remaining two patients, no informa- cells showed speckled chromatin and absence of tion regarding their outcomes could be obtained even nucleoli. The larger cell population at times showed after searching by social security number. vesicular nuclei with a single small nucleolus (Figure 2). Nuclear molding was present in only one case. The cytoplasm, in all cases, was inconspicuous. Pathological Features The mitotic count was evaluated as low in three cases, In eight cases, the sections came from the primary moderate in one case, and high in six cases. tumors: four excisions (2–8 cm in size), three The squamous cell component occurred as discrete vacuum-assisted biopsies, and one needle biopsy. foci embedded in the undifferentiated component of For two patients, only metastatic tissues, supra- the tumor. In eight cases, this component was in the clavicular lymph node and tumor mass from right form of small microscopic foci but, in two cases, it ventricular outflow tract, were available. Two cases was prominent. Four cases displayed well-developed had slides from both primary and metastatic sites. keratin pearls. Mitotic figures were identified in the The initial pathological diagnosis at the referring squamous component in only two cases. institutions was reported as poorly differentiated In one case, the residual tumor, excised post squamous carcinoma in five cases and well-differ- chemotherapy, showed significant response in the entiated carcinoma in two cases. In one case each, mediastinum but showed prominent infiltration of thymic carcinoma (not specified), large cell undif- the adjacent lung. In the lung, foci of tumor showed ferentiated type with squamous differentiation and a peribronchial growth and were associated with non-small carcinoma with neuroendocrine feature peripheral palisading. However, glandular spaces or was recorded. cyst formation, reminiscent of basaloid carcinoma, All 10 tumors were composed of squamous was not seen. cells and undifferentiated components (Table 2). In two cases, matched primary and metastatic The transition between the two components was samples were available for review. In these cases,

Table 2 The details of the morphological features of the undifferentiated and squamous cell components are listed below

Undifferentiated Squamous component component Mitotic activity

Keratin Predominant Squamous Undifferentiated Lymphocytic Case no. H&E Necrosis Amount pearl cell type component component infiltrate

1 1 No Low Absent Large No High No 2 14 Yes Low Present Large Yes High No 3 1 Yes Low Absent Large No High No 4 2 No Low Present Large No Moderate No 5 5 Yes High Absent Large No Low Yes 6 3 No High Present Large No Low Yes 7 2 Yes Low Absent Large No High No 8 3 Yes Low Present Small No Low Yes 9 1 Yes Low Absent Large Yes High Yes 10 1 No Low Absent Large No High Yes

Abbreviation: H&E, hematoxylin and eosin.

Modern Pathology (2014) 27, 1649–1656 NUT midline carcinomas YGo¨kmen-Polar et al 1653

Figure 1 Mixed small cell squamous carcinoma of the thymus. Low and high magnification images (a, b) showing the two components juxtaposed to each other with abrupt transition.

Figure 2 Mixed small cell squamous carcinoma of the thymus. Figure 3 Mixed small cell squamous carcinoma of the thymus. Undifferentiated component is predominant and the cells have Section is taken from the brain metastases in one of the patients. vesicular nuclei with small nucleoli and lack the classic molding Note that mixed pattern is retained in the metastases. of small cell carcinoma. the tumor at both sites had identical morphology Expression was also analyzed in 32 cases without (Figure 3). these histological features; none showed expression Immunohistochemistry data were available in of NUTM1 protein. seven cases, all tumors were positive for a broad spectrum of cytokeratins (data not shown). Neuro- endocrine features (synaptophysin and/or neuron- Discussion specific enolase) were also reported as positive in two of the five cases analyzed (Cases 3 and 7). The diagnosis of thymic carcinoma relies heavily on CD5 expression was positive in all three tumors clinical findings, imaging studies and supported by analyzed. histologic findings. Caution is required in making this diagnosis in the absence of appropriate context. NUTM1 Expression Analysis This is especially true when one is dealing with biopsy specimens; in all of the cases presented here, Immunohistochemistry was performed using the the possibility of extrathymic origin was excluded NUTM1 antibody (C52; Cell Signaling) gene- on clinical and imaging studies. Importantly, in all rated by French et al.11,12 Expression was noted in cases, the diagnosis from the referring institution two of the three cases with blocks (Figure 4a–c). was thymic carcinoma.

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bladder.13,14 The older articles focus on whether the undifferentiated component and squamous component had different origins (endodermal versus neuroectoderm). The characterization of the undifferentiated component is a more difficult issue. The cells, although undifferentiated, lack the classic features of small cell undifferentiated carcinoma seen in the lung. The nuclei are larger and lack nuclear molding. DNA encrustation of stroma and vessels (Azzopardi effect) was not identified in any of the cases. The lack of classic features of small cell carcinoma was also noted in the original description of the entity.6 The organoid growth pattern in many cases would suggest a neuroendocrine differentiation. However, only two of the five cases stained for the neuroendocrine markers showed positivity (as per report). In the remainder of the cases, due to lack of unstained material, the neuroendocrine differentiation could not be confirmed. Kuo et al7 studied 22 thymic carcinomas for the presence of neuroendocrine cells and identified them in 4 of 10 squamous cell carcinomas, and in 7 of 8 adeno- squamous carcinomas; in almost all cases, this population constituted less than 1% of the tumor. Wick and Ritter15 showed a relatively common occurrence of mixed neuroendocrine cell and squamous cell carcinoma of thymus, postulating the existence of a common thymic epithelial pre- cursor as the progenitor of thymic neuroendocrine cells. As illustrated in this series, these tumors were most commonly misdiagnosed as some variant of squamous cell carcinoma. Keratinizing variant of squamous cell carcinoma is composed of large polygonal cells arranged in groups and cords, nuclei are vesicular or hyperchromatic, with easily seen nucleoli. Cytoplasm is eosinophilic and keratin pearls, although sometimes focal, are present. Non- keratinizing squamous cell carcinoma often shows cohesive cellular nests with cells exhibiting distinct cell membranes and at least a moderate degree of cytological atypia. Small cell variant of squamous cell carcinoma tends to exhibit cells with more vacuolated nuclei and visible nucleoli and lack evidence of neuroendocrine differentiation. Basa- loid variant of thymic carcinoma tends to have extensive glandular or cyst-like spaces and exhibit peripheral palisading.6,16,17 Poorly differentiated Figure 4 NUTM1 expression in mixed small cell squamous squamous cell carcinomas consist of sheets or carcinoma of the thymus. The three cases with blocks showed nests of large polygonal cells with prominent strong expression (a), moderate expression (b) or no expres- nucleoli and a moderate amphophilic amount sion (c). of cytoplasm. Small cell neuroendocrine carcinoma is an important consideration, particularly if the squamous component is small and not appreciated. Mixed small cell undifferentiated squamous cell The expression of neuroendocrine markers such as carcinomas in the thymus were first described by synaptophysin, chromogranin, CD56 and neuron- Snover et al6 and more recently by Mukai et al.10 specific enolase is valuable in the distinction. However, tumors with similar morphology have Entrapped thymic remnants (Hassall corpuscles) been described in a number of other sites inclu- within the tumors can be a confounding factor. ding lung, upper respiratory tract and urinary Primary mixed thymic tumor also has to be

Modern Pathology (2014) 27, 1649–1656 NUT midline carcinomas YGo¨kmen-Polar et al 1655 distinguished from a lung primary and metastases make a definitive statement in this regard. We from other sites. recommend that the expression of NUTM1 protein The association of these tumors with carcinoma should be analyzed in cases with poorly differen- with t(15;19) translocation needs closer examina- tiated or mixed squamous undifferentiated carcino- tion.17,18 Translocation carcinomas can have very ma histology to exclude the possibility of NUT similar histology. Recently, these tumors have been midline carcinoma. shown to have NUTM1 gene fusion; this can be analyzed by cytogenetic studies or specific probes that detect the fusion.12,18 More recently, a mono- Disclosure/conflict of interest clonal antibody directed against the fusion protein has been generated. We found expression of NUT The authors declare no conflict of interest. protein in two of the three cases with available blocks and in none of the 32 cases of thymic carcinomas that lacked these histological features. References The NUT antibody is known to be associated with 100% specificity but relatively low sensitivity 1 Rosai J. Histological typing of tumours of the 8 thymus. WHO International Classification of Tumours. (87%). Given this knowledge, it is very likely that Springer: Berlin Heidelberg, 1999. the tumors described herein could be part of the 2 Blumberg D, Burt ME, Bains MS, et al. Thymic NUT midline tumor family. Classically, NUT tumors carcinoma: current staging does not predict prognosis. have been described in the pediatric age group. J Thorac Cardiovasc Surg 1998;115:303–308; discus- However, the International NUT Midline Carcinoma sion 8-9. Registry recently presented data on 63 patients with 3 Ogawa K, Toita T, Uno T, et al. 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Curr Diagn Pathol blocks in a number of cases makes it difficult to 2002;8:102–112.

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